The present invention relates to electrophoretic separation systems for the analysis of bio-molecules, such as nucleic acids. More particularly, this invention relates to a multi-channel capillary electrophoresis device and method wherein the distortion of a sample zone exiting from the end of a channel is controlled thereby resulting in enhanced detectability of such sample zone.
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Electrophoretic separations of bio-molecules are critically important in modern biology and biotechnology, comprising an important component of such techniques as DNA sequencing, protein molecular weight determination, genetic mapping, and the like. A particularly preferred electrophoresis format is capillary electrophoresis (CE), where the electrophoresis is performed in a channel, such as a capillary tube or a groove in a microfabricated chip, wafer or plate, having a small internal diameter. Capillary electrophoresis results in enhanced separation performance over traditional slab-based formats because the superior ability of the narrow-bore capillary to tolerate resistive heating allows for high electrical fields to be employed thereby resulting in fast separations in which sample diffusion is minimized.
In traditional CE systems, detection of a sample subsequent to separation is performed during electrophoresis while the sample is still located inside the channel (referred to as xe2x80x9con-channelxe2x80x9d detection). Thus, in a common capillary tube arrangement, any excitation light required to excite the sample and any emission light coming from the sample must be transmitted through the wall of the capillary tube. A drawback of this approach is that the fused silica capillary tubes often used in CE have numerous surfaces to reflect or scatter light. Problems associated with light scattering are particularly problematic when it is desired to detect fluorescence from samples located in a plurality of closely-spaced capillary tubes by fluorescence because the scattered emmission light from one capillary tube can interfere with the detection of samples in neighboring capillary tubes.
One approach to solving the problem of on-channel detection has been to detect a sample after the sample emerges from the capillary (referred to as xe2x80x9coff-channelxe2x80x9d detection). In one type of arrangement, such off-channel detection takes place in a detection cell positioned downstream of the capillary tube outlets. Typically, the detection cell is configured to exhibit superior optical characteristics, e.g., a flat quartz chamber. In one class of these systems, a xe2x80x9csheath flowxe2x80x9d of liquid is used to transport the sample from the outlet of the CE capillary tube to a detection zone at which detection of the sample takes place (Takahashi; Dovichi). A drawback of sheath flow systems is that, in order to avoid distortion of a sample zone in the detection cell, precise control of the flow rate of the sheath flow liquid is required. A second drawback of sheath flow systems is that the pressure used to drive the flow of the sheath flow liquid can cause back flow of the separation medium in the separation capillary tube, thereby negatively impacting resolution.
In another class of off-channel detection systems, a sample zone is transported from the outlet of a CE capillary tube to a detection zone located in a detection cell by electrophoresis under the influence of the same voltage difference used to conduct the electrophoretic separation (Takahashi). However, because of the larger cross-sectional area within the detection cell as compared to the lumen of the capillary tube, the electric field diverges at the capillary tube outlet causing a distortion of the sample zone as it enters and traverses the detection zone. Unchecked, such distortion can result in a severe loss of spatial resolution between adjacent sample zones exiting a single capillary tube and/or between sample zones exiting adjacent capillary tubes. This loss of spatial resolution tends to reduce the detectability of neighboring sample zones.
Generally, the present invention relates to a device and method for electrophoretically transporting a sample zone from an electrophoresis channel, via an outlet end thereof, into a detection zone or chamber downstream of the channel, where the distortion of the sample zone is controlled in a fashion permitting enhanced detectability.
The various embodiments of the device and method of the present invention find particular application in automated polynucleotide sequencing systems employing fluorescence detection and a plurality of separation channels (e.g., capillary electrophoresis tubes or microfabricated (e.g., etched) channels in a plate).
More particularly, in one of its aspects, the present invention relates to an analyte separation device, such as a CE tube or plate device, including (i) a plurality of separation channels, with each channel comprising an inlet end and an outlet end; (ii) a detection zone proximate the outlet ends; and (iii) at least one excitation-beam pathway extending through at least a portion of said detection zone. In an embodiment of the device, two or more of the channels have a variation region, in which the channel cross-sectional area varies (e.g., progressively increases), in the vicinity of (i.e., near and/or along) their outlet ends.
The device can further include an excitation-beam source for directing an excitation beam along said excitation-beam pathway(s). Any suitable beam source can be employed. In an embodiment of the invention, the beam source is a laser. The present invention contemplates for example (without limitation): a side-entry beam arrangement, a scanning or fanned beam (or other broad) illumination arrangement, and/or an up-channel (axial) illumination arrangement. In an embodiment of the latter, sample excitation takes place at, or not far beyond, the variation region of each channel.
In another of its aspects, the present invention relates to an analyte separation device, such as a CE tube or plate device, having an off-channel detection arrangement. In one embodiment, the device includes (i) a plurality of separation channels, each channel comprising an inlet end and an outlet end; (ii) a detection chamber, or zone, proximate the outlet ends; and (iii) an excitation-beam pathway extending through at least a portion of the detection chamber, with the pathway (a) being located on a side of the outlet ends opposite the inlet ends (i.e., downstream of the outlet ends) and (b) extending along a plane defined by the channels (e.g., a side-entry arrangement). Two or more of the channels are provided with a variation region, in which the channel cross-sectional area varies, along a region near respective outlet ends.
According to one embodiment, the cross-sectional area of the variation region increases along a direction extending from the inlet end to the outlet end. In another embodiment, the cross-sectional area of the variation region decreases along a direction extending from the inlet end to the outlet end. In a further embodiment, the variation region comprises a first portion in which the cross-sectional area decreases along a direction extending from the inlet end to the outlet end, and a second portion in which the cross-sectional area increases along said direction, with the second portion being immediately proximate and extending to the outlet end.
The variation region of at least two of the channels is preferably located in the vicinity of the outlet end. In one embodiment, the variation region is formed within a portion of the channel extending from the channel""s outlet end towards its inlet end, along no more than 40% of the channel length. That is, the variation region is disposed in a downstream region of the channel, no more than 40% of the channel length away from the channel""s outlet end. For example, in a 100 mm channel, the variation region would be located along the 40 mm closest to the outlet end. In other embodiments, the variation region is formed within a portion of the channel extending from the channel""s outlet end towards its inlet end along no more than 30%, 20%, 15%, 10%, 5%, and/or 3% of the channel length. That is, in these embodiments, the variation region is disposed in a downstream region of the channel, no more than 30%, 20%, 15%, 10%, 5%, and/or 3%, respectively, of the channel length away from the channel""s outlet end.
One embodiment further provides a detector disposed to detect fluorescence emitted from an observation region, within the detection chamber, whereat the excitation-beam pathway intersects an imaginary axial extension of one of the channels. In a related embodiment, the detector is configured to simultaneously detect fluorescence emitted from a plurality of such observation regions, with the observation regions being spaced apart from one another. A further embodiment includes a plurality of crosstalk zones, with each crosstalk zone being located approximately midway between a respective pair of adjacent observation regions; wherein the detector is configured to disregard (i.e., not to detect) fluorescence emitted from any such crosstalk zone.
In certain embodiments of the invention, the only structure permitting fluid communication with an upstream region of the detection chamber are separation channel discharge ends. In these embodiments, no substances are required to pass along the direction of sample migration into an upstream region of said detection zone other than substances passing therein via said channel discharge ends. Thus, in these embodiments, no sheath flow liquid need be utilized.
Another aspect of the present invention relates to an analyte separation device, comprising (i) a plurality of separation channels, with each channel comprising an inlet end and an outlet end; (ii) a detection chamber proximate the outlet ends; (iii) an excitation-beam pathway extending through at least a portion of the detection chamber, with the pathway being located on a side of the outlet ends opposite the inlet ends and extending along a plane defined by the channels; and (iv) barrier structure interposed between adjacent pairs of the channels and defining, at least in part, sidewalls bounding at least a portion of such channels; with the barrier structure having an end region in the vicinity of the terminal outlets that is tapered.
According to one embodiment, the tapered region progressively narrows toward the terminal end of the barrier structure. The terminal end of the barrier structure can, for example, form a point (e.g., it can have a substantially V-shaped cross section, taken along a plane defined by the channels). According to another embodiment, the terminal end of the barrier structure is blunt. The terminal end of the barrier structure can, for example, have a U-shaped cross-section (taken along a plane defined by the channels).
In one embodiment, the tapered region includes a first portion that tapers gradually, along a direction from the inlet end to the outlet end, and a second portion, more sharply tapered than the first portion, which begins after the first portion and extends to a terminal end of the barrier structure.
In another embodiment, the tapered region includes a first portion that gradually expands, along a direction from the inlet end to the outlet end, and a second portion, which begins after the first portion, that narrows to a terminal end of the barrier structure.
One embodiment further includes a detector disposed to detect fluorescence emitted from an observation region, within the detection chamber, whereat the excitation-beam pathway intersects an axial extension of one of the channels. In a related embodiment, the detector is configured to simultaneously detect fluorescence emitted from a plurality of such observation regions, with the observation regions being spaced apart from one another. Another embodiment further includes a plurality of crosstalk zones, with each crosstalk zone being located midway between a respective pair of immediately adjacent observation regions; wherein the detector is configured to disregard fluorescence emitted from any such crosstalk zone.
In another of its aspects, the present invention relates to an analyte separation device, comprising (i) a plurality of separation channels, each channel comprising an inlet end and an outlet end; (ii) a detection chamber proximate the outlet ends; (iii) an excitation-beam pathway extending through at least a portion of the detection chamber, with the pathway being located on a side of the outlet ends opposite the inlet ends and extending along a plane defined by the channels. The detection chamber, according to one embodiment, has a cross-sectional area, taken along a plane normal to the plane defined by the channels, within a range of from 50% to 400% of the sum of the cross-sectional areas of all of the channels, wherein the channel cross-sectional area employed in calculating such sum is the cross-sectional area predominating or prevailing along most of the channel length.
In certain embodiments, the detection chamber cross-sectional area is within a range of from 65% to 300%, a range of from 70% to 250%, a range of from 75% to 200%, a range of from 80% to 150%, a range of from 90% to 110%, a range of from 95% to 105%, and/or a range of from 98% to 102% of the sum of the cross-sectional areas of all of the channels. In one embodiment, the detection chamber cross-sectional area is equal to the sum of the cross-sectional areas of all of the channels.
One embodiment further includes a detector disposed to detect fluorescence emitted from an observation region, within the detection chamber, whereat the excitation-beam pathway intersects an axial extension of one of the channels. In a related embodiment, the detector is configured to simultaneously detect fluorescence emitted from a plurality of such observation regions, with the observation regions being spaced apart from one another. A further embodiment includes a plurality of crosstalk zones, with each crosstalk zone being located midway between a respective pair of immediately adjacent observation regions; wherein the detector is configured to disregard fluorescence emitted from any such crosstalk zone.
Still a further aspect of the present invention relates to an analyte separation device, comprising (i) a plurality of separation channels, each channel comprising an inlet end and an outlet end; (ii) a detection chamber proximate the outlet ends; (iii) an excitation-beam pathway extending through at least a portion of the detection chamber, with the pathway being located on a side of the outlet ends opposite the inlet ends and extending along a plane defined by the channels. A detector is disposed to simultaneously detect fluorescence emitted from a plurality of spaced-apart observation regions within the detection chamber, with each of the observation regions being located whereat the excitation-beam pathway intersects an axial extension of a respective one of the channels. Further included are a plurality of crosstalk zones, with each crosstalk zone being located midway between a respective pair of immediately adjacent observation regions; wherein the detector is configured to disregard fluorescence emitted from any such crosstalk zone.
In one embodiment, two or more of the channels have a variation region, in which the channel cross-sectional area varies, near the outlet end.
In another embodiment, the detection chamber has a cross-sectional area, taken along a plane normal to the plane defined by said channels, within a range of from 50% to 400% of the sum of the cross-sectional areas of all of the channels.
Another aspect of the present invention provides a method for reducing distortion of a sample zone (typically taking the form of a xe2x80x9cbandxe2x80x9d) upon exiting an electrophoretic channel containing a separation medium, comprising (a) loading a sample into the channel at an inlet end thereof (e.g., a sample-loading region); (b) applying an electric field along the channel in a manner effective to cause the sample to migrate through the medium and resolve into one or more sample zones; (c) changing the cross-sectional area of each sample zone as the sample zone passes along the vicinity of an outlet end of the channel (the cross-sectional area being taken along a plane perpendicular to the direction of sample zone travel); (d) ejecting the sample zone from the channel via the outlet end; and (e) analyzing the sample zone for the presence of one or more analytes of interest.
In one embodiment, step (e) is performed after step (d).
According to one embodiment, the method is performed on a plurality of samples in parallel, employing a plurality of channels.
In one embodiment, the channels are disposed side by side (e.g., in a linear array).
In one embodiment of the method, step (e) comprises (i) directing an excitation beam along a beam pathway which is located downstream of the channels and which extends along a plane defined by the channels; and, (ii) simultaneously detecting for fluorescence emitted from a plurality of spaced-apart observation regions, each of the observation regions being located whereat the excitation-beam pathway intersects an axial extension of a respective one of the channels. In a related embodiment, step (e) further comprises (iii) disregarding (ignoring) fluorescence emitted from any one or more of a plurality of crosstalk zones, wherein each crosstalk zone is located midway between a respective pair of immediately adjacent observation regions.
Still a further aspect of the present invention relates to a method for reducing distortion of sample zones (e.g., bands) upon exiting electrophoretic channels containing a separation medium, comprising (a) loading a plurality of separation channels with respective samples; (b) applying an electric field along the channels in a manner effective to cause the samples to migrate through the medium and resolve into one or more sample zones; (c) transferring each sample zone from its respective channel into a detection zone or chamber having a cross-sectional area, taken along a plane perpendicular to the direction of sample zone travel, within a range of from 50% to 400% of the sum of the cross-sectional areas of all of the channels.
One embodiment of the method further comprises (d) directing an excitation beam along a beam pathway that traverses at least a portion of the detection chamber, downstream of the channels, and extends along a plane defined by the channels; and (e) simultaneously detecting for fluorescence emitted from a plurality of spaced-apart observation regions, each of the observation regions being located whereat the excitation-beam pathway intersects an axial extension of a respective one of the channels.
A further embodiment of the method further comprises, while performing (e), (f) disregarding fluorescence emitted from any one or more of a plurality of crosstalk zones, wherein each crosstalk zone is located midway between a respective pair of immediately adjacent observation regions.
In another of its aspects, the present invention relates to a method for analyzing a plurality of sample zones (e.g., bands) upon exiting electrophoretic channels containing a separation medium, comprising: (a) directing an excitation beam along a beam pathway which is downstream of the channels and which extends along a plane defined by the channels; (b) simultaneously detecting for fluorescence emitted from a plurality of spaced-apart observation regions, each of the observation regions being located whereat the excitation-beam pathway intersects an axial extension of a respective one of the channels; and (c) disregarding fluorescence emitted from any one or more of a plurality of crosstalk zones, wherein each crosstalk zone is located midway between a respective pair of immediately adjacent observation regions.
In one embodiment of the method, (b) and (c) are carried out simultaneously.
A further aspect of the present invention relates to an analyte separation device, comprising (i) an array of elongate channels through which one or more samples are intended to migrate under the influence of a motive force (e.g., voltage) along the channels, with each channel including a sample-loading region and a terminal outlet downstream of the sample-loading region; (ii) a detection chamber downstream of the channel outlets, with each of the outlets communicating a respective channel with the detection chamber; and wherein each of the channels defines in part a sample-migration pathway extending longitudinally along such channel, passing through a respective outlet and traversing at least a portion of the detection chamber; and (iii) an unobstructed, excitation-beam pathway extending through the detection chamber, along which an excitation beam of light may be directed so as to simultaneously intersect, in the detection chamber, plural sample-migration pathways.
Two or more of the channels can be provided with a flow cross-sectional area (i.e., a cross-sectional area cutting through the channel along a plane normal to the direction of sample migration) that varies along a region near the terminal outlet. In one embodiment, the flow cross-sectional area increases along the direction of sample migration in the varying region. In another embodiment, the flow cross-sectional area decreases along the direction of sample migration in the varying region. In a further embodiment, in the varying region, the flow cross-sectional area includes a portion that decreases along the direction of sample migration, and a portion that increases along the same direction, the latter being proximate and extending to the terminal outlet.
In one embodiment, the detection chamber accommodates an electrode (e.g., an anode) toward which the sample zones migrate.
Another aspect of the present invention relates to an analyte separation device, comprising (i) an array of elongate channels through which one or more samples are intended to migrate under the influence of a motive force across the channels, with each channel including a sample-loading region and a terminal outlet downstream of the sample-loading region; (ii) a detection chamber downstream of the channel outlets, with each of the outlets communicating a respective channel with the detection chamber; wherein each of the channels defines in part a sample-migration pathway extending longitudinally along such channel, passing through a respective outlet and traversing at least a portion of the detection chamber; (iii) an unobstructed, excitation-beam pathway extending through the detection chamber, along which an excitation beam of light may be directed so as to simultaneously intersect, in the detection chamber, plural sample-migration pathways; and barrier structure interposed between adjacent pairs of the channels and defining, at least in part, sidewalls of such channels; with the barrier structure having an elongate terminal end region near the terminal outlets that is tapered.
In one embodiment, the tapered region progressively narrows to the terminal end of the barrier structure.
In another embodiment, the tapered region includes a first portion that tapers gradually along the direction of sample migration, and a second portion, more sharply tapered than the first portion, which begins after the first portion and extends to a terminal end of the barrier structure.
Still a further aspect of the present invention relates to an analyte separation device, comprising (i) an array of elongate channels through which one or more samples are intended to migrate under the influence of a motive force across the channels; each channel including a sample-loading region and a terminal outlet downstream of the sample-loading region, and having a substantially uniform flow cross-sectional area along its length; (ii) a detection chamber downstream of the outlets of the channels, with each of the outlets communicating a respective channel with the detection chamber; wherein each of the channels defines in part a sample-migration pathway extending longitudinally along such channel, passing through a respective outlet and traversing at least a portion of the detection chamber; and (iii) an unobstructed, excitation-beam pathway extending through the detection chamber, along which an excitation beam of light may be directed so as to simultaneously intersect, in the detection chamber, plural sample-migration pathways. The detection chamber, in one embodiment, has a cross-sectional area (taken along a plane normal to the direction of sample migration) within a range of from 50% to 250% of the sum of the flow cross-sectional areas of all of the channels.
In one embodiment, the detection zone cross-sectional area is approximately equal to the sum of all of the separation-zone cross-sectional areas.
In another of its aspects, the present invention provides an analyte separation device including a plurality of separation channels and a post-channel detection chamber; the device comprising: (a) means for causing samples loaded at an inlet end of the device to migrate through a medium held in the channels, to thereby resolve the samples into one or more sample zones; (b) means for changing the cross-sectional area of each sample zone as the sample zone passes along the vicinity of an outlet end of a respective one of the channels (the cross-sectional area being taken along a plane perpendicular to the direction of sample zone migration); and (c) means for interrogating sample zones in the detection chamber for the presence of one or more analytes of interest.
These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.